Serial intracerebral passage of dengue type 1 and type 2 viruses in mice was shown previously to attenuate these viruses for humans. Dengue type 4 virus strain H241 was also successfully adapted to replicate in mouse brain. The mouse-adapted DEN4 H241N was highly neurovirulent, whereas its parent DEN4 H241P was not. In addition, DEN4 H241N replicated less efficiently than DEN4 H241P in simian LLC-MK2 cells. An intratypic DEN4 chimera containing the C-PreM-E structural protein genes from DEN4 H241N also exhibited marked restriction of growth in LLC-MK2 cells. Analysis of viral proteins produced in LLC-MK2 cells by DEN4 H241N or its derived C-PreM-E chimera indicated that very little PreM was produced and that which was detected migrated slightly slower than the PreM of DEN4 H241P or its chimeric derivative. Recent evidence indicates that immature PreM-containing flaviviruses replicate less efficiently than the mature M-containing virus. Studies were performed to determine whether the altered PreM or mutations in C or E might affect the normal processing of PreM to produce M normally present in the mature virion. Protein analysis indicated that DEN4 E, PreM, M and C were detected in the virion preparation of DEN4 H241P or its derived chimera. On the other hand, the virion preparation of DEN4 H241N or its derived chimera contained E, PreM and C, but M was not detected. This suggested that cleavage of PreM to M was defective for DEN4 H241N and the genetic loci for the defect mapped within the C-PreM-E genes. There were six amino acid differences in the structural protein gene region between DEN4 H241P and DEN4 H241N: 1 in C, 2 in PreM and 3 in E. To identify mutations responsible for the defective PreM cleavage, 8 chimeric mutants were constructed that contained one or more amino acid substitutions that are present in the mutant C, PreM or E. Only mutant DEN4(H241P, S456) which contained all three amino acid substitutions in E exhibited the PreM cleavage defect. Interestingly, chimeric mutants which contained both mutations in PreM processed PreM normally. This suggests that PreM interacts with E during virus maturation.